Open AccessEvaluation of nonisotopic labeling and detection techniques for nucleic acid hybridization
Author(s) -
Diamandis Eleftherios P.,
Hassapoglidou Stavroula,
Bean Courtney C.
Publication year - 1993
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1860070308
Subject(s) - digoxigenin , microbiology and biotechnology , nucleic acid , southern blot , dig , streptavidin , hybridization probe , genomic dna , biotin , dna , chemistry , biology , blot , gene , biochemistry , in situ hybridization , gene expression
We have used a double‐stranded DNA probe linked to the cystic fibrosis locus to detect a single‐copy gene from varying amounts of genomic DNA, with Southern blot analysis. The DNA plasmid probe was labeled with either biotin or digoxigenin. Biotin or digoxigenin was then linked to alkaline phosphatase (ALP) with use of streptavidin or anti‐digoxigenin antibodies, respectively. ALP activity was then detected with a chromogenic (BCIP/NBT), chemiluminogenic (AMPPD), or fluorogenic (HNPP) substrate. Our results suggest that biotin and digoxigenin perform similarly and that the three substrates exhibit similar detectability under appropriate substrate incubation times: 20‐120 min (AMPPD), 2‐12 h (HNPP), and 18‐48 h (BCIP/NBT). Under optimised conditions and the probe used, these methods detect single‐copy genes from as little as 0.3 μg of total genomic DNA. © 1993 Wiley‐Liss, Inc.